Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.     

Highly sensitive and fast detection of Phoma tracheiphila by polymerase chain reaction

Summary A new method for the diagnosis of the plant pathogenic fungus Phoma tracheiphila has been developed. The method takes advantage of the enzymatic amplification of a specific 102 bp-long target sequence of fungal DNA by the polymerase chain reaction (PCR) using Thermus aquaticus DNA polymerase. The amplified DNA was characterized by agarose-gel electrophoresis, molecular hybridization using a synthetic oligonucleotide probe and direct sequencing. The application of the new method makes possible fast and direct detection of the pathogen in lignified plant tissues, a goal not previously achieved when a cloned probe and a dot-blot test were employed. In addition the PCR test can be used to advantage as a particularly simple and fast way of typing fungal isolates. This is achieved by submitting to DNA amplification crude homogenates of fungal mycelium and analysing the amplified DNA on an agarose mini-gel.Offprint requests to: F. Rollo     

Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.     

Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits.

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.     

Polypeptides of DNA-dependent RNA polymerase of spinach chloroplasts: characterization by antibody-linked polymerase assay and determination of sites of synthesis

Using solid-phase `Sandwich' immunoassays we studied DNA-dependent RNA polymerase of spinach chloroplasts with regard to (i) polypeptide composition of the multimeric enzyme; (ii) immunological cross-reaction with Escherichia coli RNA polymerase; (iii) sites of synthesis of polymerase polypeptides. Our main results are as follows. (i) All polypeptides of isolated chloroplast RNA polymerase (150, 145, 110, 102, 80, 75 and 38 kd) are labeled by an antibody-linked polymerase assay (ALPA), i.e., they are immunologically related to subunits of the holoenzyme. On the other hand differences in the patterns of `ALPA-reactive' polypeptides of a crude RNA polymerase fraction and of the purified enzyme preparation indicate partial proteolytic degradation of polymerase polypeptides during purification. Thus the 80- and 75-kd polypeptides, which had been previously considered as true RNA polymerase polypeptides, probably result from partial proteolytic degradation. (ii) The 150- and 145-kd polypeptides show immunochemical similarities with the β and/orβ' subunits of E. coli RNA polymerase. (iii) Results from solidphase immunoassay of in vitro translated products of both chloroplast RNA and poly(A)⁺ (nuclear) RNA suggest that all chloroplast RNA polymerase polypeptides are coded for by the nucleus.     

Calcineurin: a member of a family of calmodulin-stimulated protein phosphatases

Calcineurin, a major calmodulin-binding protein of brain, is a heterodimer composed of a 61,000 Mr calmodulin-binding subunit, calcineurin A, and a 19,000 Mr Ca2+-binding subunit, calcineurin B. The discovery of a calmodulin-regulated protein phosphatase in rabbit skeletal muscle with a similar subunit structure led to the identification of calcineurin as a protein phosphatase (AA Stewart, TS Ingebritsen, A Manalan, CB Klee, P Cohen (1982) FEBS Lett 137:80-84). Using rabbit polyclonal antibodies to bovine brain calcineurin, both subunits of calcineurin can be identified in crude homogenates of bovine brain by an immunoblotting technique. In crude homogenates of bovine skeletal and cardiac muscle, a 59,000-61,000 Mr doublet and a 15,000 Mr species (the electrophoretic mobility of calcineurin B) are also detected by this technique. The cross-reactivity of these species with antibodies to brain calcineurin indicates antigenic similarity between the muscle proteins and calcineurin, and suggests the existence of a family of structurally related calmodulin-stimulated protein phosphatases. Like calcineurin, the 61,000 Mr subunits in skeletal and cardiac muscle bind calmodulin and are detected in crude tissue extracts by 125I-calmodulin gel overlay. Thus, both the 125I-calmodulin gel overlay method and the immunoblotting technique are useful in screening crude preparations, in which detection of calmodulin-stimulated protein phosphatase activity may be complicated by the many phosphatases present.     

The molar ratio of σ73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii

In the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ⁷³ to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno-precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ⁷³ to core polymerase in R prowazekii would be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.     

The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential.

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.     

A simplified method for reconstituting active E. coli DNA polymerase III

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ?, θ, γ, δ′, δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be co-expressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.     

A method for assaying DNA-dependent RNA polymerase II in crude extracts of Drosophila melanogaster adults: Its use in identifying mutants with an altered RNA polymerase II

A method for assaying Drosophila melanogaster adult DNA-dependent RNA polymerase II in crude extracts from as few as two females or three males is described. Preparation of the extracts involves incubating homogenates at 25 C for 60 min and subsequent treatment with Macaloid. Eighty-five percent of the activity in the extracts is inhibited by 1 µg/ml -amanitin and this fraction is attributed to RNA polymerase II. RNA polymerase II activity in the extracts shows a good dose dependence and a partial dependence on added DNA, Mn²⁺, and all four ribonucleoside triphosphates. The kinetics of heat inactivation of RNA polymerase II in crude extracts could be reproducibly measured. Flies of different genotypes had different initial rates of RNA polymerase II heat inactivation. The isolation of Drosophila melanogaster -aminitin-resistant mutants is also reported. Using the assay described in this paper, it appears that the basis for the resistance is an altered RNA polymerase II. The mutation has been mapped to the third chromosome by chromosome replacement.Supported by Grants GM23456 from the NIH and 11259 from the City University Research Foundation.     

The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the alpha subunit

The Escherichia coli dnaE gene, which encodes the alpha subunit of DNA polymerase III (pol III) holoenzyme, has been cloned in a plasmid containing the PL promoter of phage lambda and thermally induced to overproduce the alpha subunit. In cells carrying this plasmid (pKH167), the alpha subunit was amplified, after heat induction, to a level of about 0.2% of the total cellular protein. Polymerase activity was assayed in three ways: (i) gap-filling by pol III holoenzyme and subassemblies of it, (ii) the extensive replication of a primed, single-stranded DNA circle only by pol III holoenzyme, and (iii) complementation of a crude, inactive pol III holoenzyme (temperature-sensitive dnaE mutant fraction) in replication of a primed, single-stranded DNA circle. Amplification of the alpha subunit raised the polymerase level 10-fold in assay (i), indicative of the dependence of pol III gap-filling activity on this polypeptide; pol III holoenzyme activity remained unaffected (assay (ii)), but the complementation activity was raised 5-fold (assay (iii)). Thus, the elevated alpha subunit (free or in a subassembly form) can substitute in vitro for a defective alpha subunit in pol III holoenzyme, but cannot increase the in vivo level of about eight pol III holoenzyme molecules per cell. This low level of pol III holoenzyme is fixed in wild type cells (bearing no plasmid) despite the presence of a 5-fold excess of the alpha subunit, as inferred from the various assays. These results suggest that the low level of pol III holoenzyme is determined by a factor or factors other than the level of the alpha subunit.     

DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis

Processive DNA synthesis, a property of DNA polymerase III holoenzyme of Escherichia coli, was not achieved by combining the pol III core (alpha, epsilon, and theta subunits) and the beta and gamma subunits. An activity that restored processivity to these subunits was found in crude extracts and was overproduced 4-fold in cells with plasmids amplifying the tau and gamma subunits. Purified to homogeneity, the activity, assayed by reconstitution of processivity, was represented by five polypeptides which were copurified. Judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these correspond to the known subunits gamma (52 kDa) and delta (35 kDa) and to three new polypeptides: delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The five polypeptides form a tight complex with a native molecular weight of about 200 kDa and a subunit stoichiometry of two gamma subunits to one each of the others. Processive DNA synthesis, now achieved with only three components (pol III core, beta, and the auxiliary complex), provides the opportunity to assess the functions of each and the contribution that the remaining auxiliary tau subunit makes to reconstitute a holoenzyme.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.